This invention relates to methods of measuring retinal damage. In particular, the invention relates to methods of measuring retinal damage in situ by measuring changes in retinal thickness resulting from such damage.
The human retina includes three primary nuclear layers that include five major classes of neurons. The five major classes of neurons are: (i) photoreceptors (rods and cones); (ii) bipolar cells; (iii) horizontal cells; (iv) amacrine cells; and (v) ganglion cells. The outer nuclear layer (ONL) contains the photoreceptor cell bodies. The inner nuclear layer (INL) contains the cell bodies of the bipolar neurons, horizontal neurons, and amacrine neurons. Interplexiform neurons, displaced ganglion cells, throughout the glial cells of Mxc3xcller are also located in the INL. The ganglion cell layer contains the cell bodies of most of the ganglion cells, displaced amacrine cells, and some astroglial cells.
Synaptic connections are made among the various classes of neurons, which result in the vertical and lateral flow of visual information in the retina. Both excitatory and inhibitory synaptic connections are present in the retina. Glutamate is probably the primary excitatory neurotransmitter. Gamma-aminobutyric acid (GABA) is probably a major inhibitory neurotransmitter. Other neurotransmitters are present among the various retinal neurons.
Lasers are increasingly being used in research, medical, industrial, and military fields. Eye exposure to lasers may be accidental or intended. For example, ophthalmic laser treatment is a treatment for many conditions of the eye, including age-related macular degeneration (AMD), macular edema, and photorefractive keratectomy (PRK). In addition, a number of diseases that involve macular degeneration that are potentially treated with lasers include Stargardt""s disease, Best""s disease, Batten""s disease, Sjogren-Larsson syndrome, cone-rod dystrophy, and ovine ceroid lipofuscinosise, and Tay-Sach""s disease. At least some types of AMD are caused by increased neovascularization.
Laser photocoagulation treatment utilizes thermal energy to destroy neovascular tissue. The energy of the laser heats the tissue and results in full-thickness retinal damage including secondary, collateral damage to the tissue surrounding the laser contact site. The secondary, collateral damage may result from physiological effects, such as excessive release of the neurotransmitter, glutamate, resulting from the primary damage caused by the laser (Marshall et al., xe2x80x9cHistopathology of ruby and argon laser lesions in monkey and human retina. A comparative studyxe2x80x9d, Br. J. Ophthalmol., 59(11):610-630 (1975); Rosner et al., xe2x80x9cNeuroprotective therapy for argon-laser induced retinal injuryxe2x80x9d, Exp. Eye Res., 65:485-495 (1997)). Additionally, the secondary, collateral damage may be attributed to phospholipid hydrolysis and arachidonic acid metabolites, oxygen free radicals, changes in intracellular and extracellular ion concentrations, and other excitotoxic mechanisms. The secondary damage may exacerbate the primary anatomical and physiological damage caused by the laser and may result in unwanted side effects, such as further vision loss, of the subject exposed to the laser.
Retinal damage caused by lasers is conventionally assessed ophthalmoscopically using opthalmoscopes slit lamps and/or retinal photographs (xe2x80x9cfundus photosxe2x80x9d). Using ophthalmoscopes, retinal lesions produced by lasers may be measured by viewing the diameter and intensity of the lesion(s) on the retina. Because light energy from a laser is converted into thermal energy by light absorption of the retinal pigment epithelium and choroid, the retina is damaged by the heat conduction, and the thermally-injured retina loses its transparency and scatters white light back to the observer. Greater primary damage correlates with less transparency and a whiter lesion. When fundus photos are utilized, a physician may inject a fluorescent drug (such as flouroscein) into a patient""s blood, and allow enough time for the drug to circulate throughout the patient""s blood vessels and capillaries. The physician then uses topical eye drops to dilate the pupil, and takes a magnified photograph (xe2x80x9cangiogramxe2x80x9d) of the retina, using an ultraviolet light source with a wavelength that causes the drug in the patient""s blood to emit fluorescent light at a different wavelength from the light source. Lesions are characterized by fluorescent regions corresponding to blood vessels that have been exposed resulting from the thinning or removal of the overlying retinal tissue.
Lesions can be classified in a number of ways (see, for example, Mainster, Decreasing retinal photocoagulation damage: principles and techniquesxe2x80x9d, Seminars in Ophthalmology, 14(4):200-209 (1999)). Lesions can be classified by their appearance at the time of laser treatment, ranging from faint to intense white. Lesions can also be classified by how they are observed (e.g. angiographically versus ophthalmoscopically). Lesions can also be classified by their latency from injury. In addition, retinal lesions have been examined histopathologically (Rosner, et al., Neuroprotective therapy for argon-laser induced retinal injury, Exp. Eye Res. 65:485-495 (1997); Robertson et al., Laser pointers and the human eye, A clinicopathologic study, Arch Ophthalmol., 118:1686-1691 (December 2000); Zuclich et al., Retinal damage induced by red diode laser, Health Phys., 81(1):8-14 (2001)).
Conventional in situ methods of estimating retinal damage evaluate the primary damage caused by the laser (i.e., the diameter of the lesion as seen by the xe2x80x9cwhitenessxe2x80x9d of the lesion, or by the fluorescence of the lesion). The conventional in situ methods do not measure the secondary, collateral damage resulting from laser injuries. The secondary damage caused by the laser has only been estimated histopathologically (e.g., Rosner et al., supra); however, histopathological examination is not suitable for patients.
Therefore, there remains a need for an in situ method for measuring retinal damage, including secondary retinal damage, resulting from a retinal injury. Such a method will not only provide the ability to quantify retinal damage, including secondary retinal damage, resulting from focal injuries, such as laser injuries, but will also be useful for screening pharmaceutical agents for neuroprotective effects against such damage.
The present invention meets this need and provides a method for measuring retinal damage in situ. The methods of the invention enable one to measure or quantify the amount of damage caused by retinal injury without removing an eye or eyes from a subject so injured. In addition, the methods of the invention provide an in situ method for determining neuroprotective effects of ophthalmic solutions.
In one embodiment of the invention, a method for measuring retinal damage resulting from a focal injury to a retina comprises the step of calculating a slope of retinal thickness between a plurality of locations of the retina. In one embodiment of the invention, the focal injury is caused by a laser.
In another embodiment of the invention, a method for measuring retinal damage caused by a laser comprises the steps of: (i) measuring the thickness of the retina, which has been exposed to a laser, at a plurality of locations on the retina; and (ii) calculating the slope of retinal thickness from the site exposed to the laser to an unaffected site.
In a further embodiment of the invention, a method for measuring retinal damage may comprise measuring and calculating the average retinal thickness in the laser-induced lesion area and the surrounding retina.
In practicing the foregoing methods, retinal thickness is preferably measured in situ. A greater amount of retinal damage corresponds to a more shallow slope of retinal thickness. In addition, the retinal damage measured by the foregoing methods comprises secondary damage from the focal injury. The retinal thickness may be measured after acute inflammation produced by the injury has subsided.
The foregoing methods may also comprise an additional step of administering a neuroprotective agent to a subject. The neuroprotective effects of the agent correlate with an increased slope of retinal thickness from the site of injury to the periphery of the injury as compared to the slope of retinal thickness in a subject that has not received the neuroprotective agent. In certain embodiments, the neuroprotective agent is administered before the injury, such as when a laser causes a retinal lesion. The neuroprotective agent used in the foregoing method may reduce excitotoxicity, or may reduce secondary retinal cell death resulting from the laser injury.
Alternatively, the foregoing methods may be useful for screening pharmaceutical compositions for neuroprotective effects. Such methods comprise the step of administering a potential neuroprotective agent to a subject. The neuroprotective effects of the potential neuroprotective agent correlate with an increased slope of retinal thickness as compared to the slope of retinal thickness of an injured retina of a subject that has not received a neuroprotective agent.
In still a further embodiment of the invention, a method for determining neuroprotective effects of a compound, such as a compound in an opthalmic solution, comprises the steps of: (i) administering a potential neuroprotective agent in an ophthalmic composition to a subject; (ii) measuring the thickness of a retina, which has been exposed to a laser, at a plurality of locations of the retina; and (iii) calculating the slope of retinal thickness from the lesion of the laser to the periphery of the lesion.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.
Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.